Spätzle processing enzyme is required to activate dorsal switch protein 1 induced Toll immune signalling pathway in Tenebrio molitor

Dorsal switch protein 1 (DSP1) acts as a damage-associated molecular pattern (DAMP) molecule to activate immune responses in Tenebrio molitor. From a previous study in Spodoptera exigua, we found that DSP1 activates Toll immune signalling pathway to induce immune responses by melanisation, PLA2 activity and AMP synthesis. However, the target site of DSP1 in this pathway remains unknown. The objective of this study was to determine the role of spätzle processing enzyme in the DSP1 induced toll immune signalling pathway. To address this, we analyzed spätzle processing enzyme (Tm-SPE) of the three-step serine protease cascade of T. molitor Toll pathway. Tm-SPE expressed in all developmental stages and larval tissues. Upon immune challenge, its expression levels were upregulated but significantly reduced after RNA interference (RNAi). In addition, the induction of immune responses upon immune challenge or recombinant DSP1 injection was significantly increased. Loss of function using RNA interference revealed that the Tm-SPE is involved in connecting DSP1 induced immune responses like hemocyte nodule formation, phenoloxidase (PO) activity, phospholipase A2 (PLA2) activity and antimicrobial peptide (AMP) synthesis. These suggest that Tm-SPE controls the DSP1 induced activation of Toll immune signalling pathway required for both cellular and humoral immune responses. However, to confirm the target molecule of DSP1 in three-step proteolytic cascade, we have to check other upstream serine proteases like Spatzle activating enzyme (SAE) or modular serine protease (MSP).


Introduction
High mobility group box 1 (HMGB1) is a ubiquitously expressed, highly conserved 'chromatin-associated protein' in vertebrates [1].At normal condition, it remains in nucleus to contribute gene expression, DNA repair, chromatin rearrangement etc. [2][3][4] while released from nucleus under stress to acts as a damage-associated molecular pattern (DAMP) that activate innate immune responses by interacting with pattern recognition receptors like Toll-like receptors (TLRs), receptor for advanced glycation end products (RAGE) etc. [5].Dorsal Switch Protein 1 (DSP1) is the insect homolog of vertebrate HMGB1 first introduced in Drosophila melanogaster [6] as a transcriptional co-repressor and chromatin remodelling factor [7,8].DSP1 exhibits nuclear function like DNA binding [9], binding with zen and Rel to activate transcription in Anophiles gambie [10], regulation of Sex combs reduced (Scr), Ultrabithorax (Ubx) and Abdominal-B gene expression [8].DSP1 also mediate immune responses upon pathogen infection in insects like Spodoptera exigua [11] and Tenebrio molitor [12] by activating eicosanoid that mediate cellular and humoral immune responses in insects [13].Following Toll signalling pathway, DSP1 activate phospholipase A 2 (PLA 2 ), an enzyme that catalyse phospholipids to biosynthetic precursor(s) eicosanoids [14].
Toll immune signalling pathway in Drosophila is induced by Gram-positive bacteria or fungi and begins with the recognition of lysine-type peptidoglycan (PG) that triggers activation of the serine protease (SP) cascade leading to form active Spa ¨tzle [15,16].In Tenebrio molitor, SP cascade consists of three SPs [17,18].The initiating SP is a modular serine protease (modSP) which binds to the lysine-type PG recognition complex and activates the second SP, Spa ¨tzle activating enzyme (SAE), which in turn activates the last SP, Spa ¨tzle processing enzyme (SPE) [17,19].Inactive pro-Spa ¨tzle is cleaved by SPE to form active Spa ¨tzle, which binds to Toll receptor and triggers its specific immune signalling pathway [20,21].SPE also cleaves inactive prophenoloxidase (PPO) to active phenoloxidase (PO) [22] that leads to melanisation on the surface of bacteria to kill them.Thus, SPE mediates Toll immune signalling pathway to produce antimicrobial peptides (AMPs) and the melanisation immune responses [19].
DSP1 was predicted and known to mediate immune responses in T. molitor [12] and showed that DSP1 up-regulated gene expression of some AMPs and activated PLA 2 , PO and nodulation in response to Gram-positive bacterium containing Lys-type PG.A previous study in S. exigua reported that DSP1 activate Toll immune signaling pathways for immune mediation [14].Another study in T. molitor reported that the Toll pathway includes a three-step proteolytic cascade such as modSP, SAE and SPE for mediating immune responses induced by peptidoglycans [17].But we do not know the role of SPE or other upstream serine protease in DSP1 induced Toll pathway.Thus, present study aimed to know the involvement of SPE among the three-serine protease of T. molitor Toll pathway in mediating DSP1 induced immune responses.Present study showed that SPE control the immune responses induced by DSP1.This suggests that DSP1 depends on the SPE to activate Toll immune signalling pathway in T. molitor.

Chemicals
L-3,4-dihydroxyphenylalanine (DOPA) was purchased from Sigma-Aldrich Korea (Seoul, Korea) and dissolved in 100 mM PBS before assay.Anticoagulant buffer (ACB) needed to prevent hemocyte coagulation was prepared with 186 mM NaCl, 17 mM Na 2 EDTA and 41 mM citric acid adjusting the pH at 4.5.Metafectene Pro, a transfection reagent, needed to mix with dsRNA was purchased from Biontex (Plannegg, Germany).Phosphoric acid (100 mM) was used to prepare phosphate-buffered saline (PBS, pH 7.4).Purified recombinant DSP1 (rDSP1) of Spodoptera exigua was prepared in a previous study [11] and used in this study.

Bioinformatics and sequence analysis
Tm-SPE was predicted from the whole genome shotgun contig (GenBank accession number: JABDTM010027791.1)using the Drosophila SPE as bait.The resulting sequence was subjected to further analysis with FGENESH Soft berry program (http://www.softberry.com/berry.phtml) to obtain an open read frame (ORF).ORF sequences were deposited to GenBank (accession number: MZ 190162.1).Phylogenetic analyses were performed using MEGA6 and Clustal W programs from EMBL-EBI (www.ebi.ac.uk) using Neighbor-joining method and the used genes accession numbers are presented in S2

RNA extraction and RT-qPCR
Total RNAs from different developmental stages were extracted using 50 eggs, 20 L1 larvae, 10 L2 larvae, 2 L6 larvae, 1 L12 larva, one pupa, and one adult per replication.To extract total RNAs from different tissues, L6 larvae were used.Hemolymph from larvae was collected in ACB by cutting the abdominal tip.The collected hemolymph was centrifuged at 500 × g for 5 min.The resulting hemocyte pellet was used to extract total RNA.The remaining body after hemolymph collection was used to isolate fat body, midgut, and epidermis.Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used for total RNAs extraction.After DNase treatment, 1 μg of total RNA was used to synthesize first-strand cDNAs using RT-Premix oligo-dT following [12].Resulting cDNAs were used for PCR amplification with gene-specific primers (S1 Table ).RT-qPCR was conducted using a Step One Real-Time PCR System (Applied Biosystem, Marsiling, Singapore) following the conditions: 95˚C for 10 min for initial heat followed by 40 cycles of 95˚C for 30 sec, different annealing temperature for 30 sec and 72˚C for 30 sec.T. molitor specific gene, L27a, was used as reference gene [23].Quantitative analysis was done with a comparative CT method [25] using the reference gene for normalization to estimate mRNA expression levels.Each treatment was replicated three times.Each time with three independent samples.

RNA interference (RNAi) of Tm-SPE expression
Gene-specific primers (S1 Table) containing T7 promoter sequence (5´-TAATACGACTCAC TATAGGGAGA-3´) at the 5´ends were used to amplify template DNA.The resulting PCR product was used to synthesize double-stranded RNA (dsRNA) encoding Tm-SPE (dsSPE) using a MEGAscript RNAi kit (Ambion, Austin, TX, USA) at 37˚C for 4 h.Synthesized singlestranded RNAs in both directions were allowed to anneal at 25˚C after heat treatment at 75˚C for 5 min.Control dsRNA (dsCON) was synthesized from a viral gene, CpBV302 [26].dsRNAs were mixed with a transfection reagent Metafectene PRO at a 1:1 (v/v) ratio and then incubated at 25˚C for 30 minutes to allow liposome formation that increases RNAi efficiency [27].One μg of dsRNA for each was injected into L6 using 10 μL Hamilton microsyringe.The RNAi efficiency was determined by RT-qPCR against Tm-SPE expression at 24, 48, 72 and 96 h post-injection (PI).

Nodulation assay
Nodule formation assay was analysed to investigate the cellular immune response against E. mundtii or rDSP1 following [28].Heat-killed E. mundtii (1.8 × 10 5 cells/larva) was injected to L6 larvae through the pleuron using 10 μL Hamilton microsyringe at 24 h of dsRNA injection to induce hemocyte nodules.Number of nodules formed by T. molitor in the hemocoel as cellular immune response against bacterial challenge were counted.To rescue RNAi effect, recombinant DSP1 (rDSP1) was injected (600 ng/larva) along with bacterial challenge.After 8h incubation at 25˚C, treated larvae were dissected under a stereomicroscope (Stemi SV 11, ZEISS, Jena, Germany).Nodules on the fat body were counted after removing the gut.Each treatment was replicated three times.Each time with five larvae.

Phospholipase A 2 (PLA 2 ) assay
Both sPLA 2 and cPLA 2 enzyme activities in treated larvae samples were analyzed as described previously [12] using PLA 2 Assay Kit (Cayman Chemical, Ann Arbor, MI, USA).For induction of PLA 2 enzyme activity, L6 larvae were injected with 2.2 × 10 5 cells of heat-killed E. mundtii.Tm-SPE was silenced using dsRNA specific to Tm-SPE by injecting 24 h before bacteria and rDSP1 injection.Hemolymph and fat body samples from treated larvae were collected at 8 h after the bacteria and rDSP1 injection.Plasma was separated from hemocyte by spinning at 500 × g for 5 min.Plasma and fat body samples were used for measuring sPLA 2 and cPLA 2 activity, respectively.Each treatment was replicated three times.Each time with three independent samples.

Phenoloxidase (PO) enzyme assays
PO activity in plasma of treated larvae was determined using DOPA substrate as described by [12].To induce PO activity, L6 larvae were challenged with E. mundtii dissolved in PBS before 8h of sample (plasma) collection.Naïve larvae were injected with sterile PBS.The total reaction volume (200 μL) consisted of 180 μL of 10 mM DOPA in PBS and 20 μL of plasma collected from treated larvae.Absorbance was measured at 490 nm using a VICTOR multi label Plate reader (PerkinElmer, Waltham, MA, USA).PO activity was expressed as ΔABS/min/μL of plasma.Each treatment was replicated three times.Each time with three independent samples.

AMP genes expression pattern
Whole body tissues were evaluated for AMP genes expression that represent humoral immune responses.AMP genes expression in L6 larvae were determined after injecting 2.2 × 10 5 cells of heat killed E. mundtii per larvae at 24 h post injection of dsRNAs.To rescue the RNAi effect, rDSP1 (600 ng/larva) was injected to the larvae along with the bacterial cells.At 8 h of bacteria injection, samples were collected to be used for RNA extraction.Three larvae were used for sample collection and subsequent RNA extraction, for each replication.AMP genes expression levels were assessed by RT-qPCR using gene specific primers (S1 Table) using L27a as reference gene.Each treatment was replicated three times.Each time with three independent samples.

Bioassay of RNAi-treated T. molitor against E. mundtii (Em) and X. hominickii (Xh)
To assess the virulence of Gram-positive E. mundtii and Gram-negative X. hominickii on RNAi-treated T. molitor, L6 larvae were injected with 1 μg of dsRNA specific to Tm-SPE along with transfection agent, metafectene at 1:1 ratio.For control, larvae were injected with control dsRNA along with metafectene.After 24 h of dsRNA injection when the Tm-SPE is mostly silenced, same larvae were injected with either E. mundtii (2.2 x 10 5 cells/larvae) or X. hominickii (1.8 x 10 5 cells/larvae) to observe their virulence against T. molitor larvae.For injection, overnight grown bacteria were spined for 5 minute and the pallets were dissolved in PBS.The vial containing the bacterial suspension was vortexed very well to confirm almost equal number of bacterial insertion using Hamilton syringe.The treated larvae were incubated at room temperature (25 ± 2˚C) providing enough diet for another 7 days under the rearing conditions and counted the mortality at each 24 h.To check the immune association of DSP1, rDSP1 was injected along with gram positive E. mundtii.Each treatment was replicated three times.Each replication consists of ten larvae.

Statistical analysis
All data for continuous variables were subjected to one-way analysis of variance (ANOVA) using PROG GLM in SAS program [29].Mortality data were subjected to arcsine transformation and used for ANOVA.Means were compared with the least significant difference (LSD) test at Type I error = 0.05.Median lethal dose (LD 50 ) was obtained from Probit analysis using EPA Probit Analysis Program, ver.1.5 (U.S. Environmental Protection Agency, USA).Each treatment was replicated three times.

Prediction of spa ¨tzle processing enzyme (Tm-SPE) from T. molitor transcriptome
Tm-SPE was predicted from a transcriptome (GenBank accession no.JABDTM010027791.1)by interrogation with D. melanogaster SPE sequence (GenBank accession no.NM_142911.4)as a query.It has 99.65 percent sequence similarity with previously predicted Tm-SPE [17] and was deposited to GenBank (Accession no.MZ 190162.1).Its ORF consists of 2217 bp encoding 738 amino acids.The predicted amino acid sequence of Tm-SPE was compared to those of other insect SPE genes through a phylogenetic analysis where SPE genes of the insects of same order clustered together (Fig 1A

Expression profile of Tm-SPE in Tenebrio molitor
Tm-SPE was expressed in all developmental stages ranging from egg to adult and showed high expression levels at larval stages, especially at L6.However, its expression levels were low at the egg and pupal stage than adult stage (Fig 2A ).At L6, it was expressed in all tested tissues including hemocytes, fat body, gut, and epidermis.However, its expression levels were different among tissues, showing a high expression level in the fat body and low expression level in epidermis (Fig 2B).Basal expression levels were significantly upregulated following immune challenge with E. mundtii in whole body tissue where the maximum expression (more than 7-fold) was at 8 h of injection (Fig 2C).However, at very early stage (2 h of injection), expression level was around 3-fold.The upregulation of Tm-SPE expression after bacterial challenge suggested its immunological function.

RNAi of Tm-SPE and interruption of nodulation and PO activity
Expression of Tm-SPE was suppressed by RNAi via injection of gene-specific dsRNA.Such RNAi treatment significantly (P < 0.05) reduced the expression levels around 60 percent at 24 h of dsRNA post injection which continued up to 48 h and then starts to increase.Such reduced levels were recovered at 96 h PI (Fig 3A).These down regulation of Tm-SPE expression denotes the efficiency of RNAi of Tm-SPE gene.
To know the immune association of Tm-SPE, nodulation and PO activity was analyzed at both non-silenced and silenced condition of Tm-SPE.At non-suppressed condition, increased number of nodules (62 ± 5.70) formed in the insect hemocoel upon E. mundtii challenge which decreased (47 ± 4.47) significantly (P < 0.05) at the Tm-SPE silenced condition obtained For both the cases, induction was very high and significantly (P < 0.05) more compared to naïve and even E. mundtii which decreased significantly (P < 0.05) at silenced condition of Tm-SPE gene (Fig 3B and 3C).This result thus denotes that both E. mundtii and rDSP1 depends on SPE for the induction of hemocyte nodulation and PO activity.

Induction of PLA 2 activity in T. molitor by E. mundtii or rDSP1
Upregulation of Tm-SPE after immune change indicate its immune association.To validate this, both sPLA 2 and cPLA 2 activity was analysed upon immune challenge.Induction of sPLA 2 activity was significant (P < 0.05) upon E. mundtii challenge which was reduced after RNAi of Tm-SPE (Fig 4A).Similarly, cPLA 2 is also induced significantly by E. mundtii but RNAi of Tm-SPE suppressed it (Fig 4B).To see the immune mediation by DSP1, recombinant DSP1 was injected into the larvae of T. molitor.rDSP1 injected larval plasma showed significantly higher sPLA 2 (Fig 4A ) and cPLA 2 activity (Fig 4B ) which was reduced significantly after RNAi of Tm-SPE gene.This result suggests that rDSP1 can activate both the sPLA 2 and cPLA 2 activity similar to E. mundtii depending on SPE for mediation of PLA 2 activity.

Effect of spa ¨tzle processing enzyme (SPE) on susceptibility of T. molitor
Toll-spa ¨tzle immune signaling pathway is activated by Gram-positive bacteria.To confirm the involvement of Tm-SPE in Toll pathway activation, we checked virulence of both Gram-positive (E.mundtii) and Gram-negative (X.hominickii) bacteria against T. molitor larvae after individual RNAi treatment with dsSPE.Control larvae were treated with dsCON.For control larvae, E. mundtii found less virulent compared to X. hominickii whereas E. mundtii showed more virulence than X. hominickii against dsSPE treated larvae (Fig 6A).This indicate that SPE of T. molitor Toll pathway is essential for immune activation in Gram positive E. mundtii expressed.A ribosomal gene, L27a, was used as internal control [24].Each treatment was replicated three times with independent samples preparation.Different letters above the standard deviation bar indicate significant differences among means at Type I error = 0.05 (LSD test).https://doi.org/10.1371/journal.pone.0291976.g002https://doi.org/10.1371/journal.pone.0291976.g003bacterium.To confirm the immune role of DSP1, recombinant DSP1 was injected into T. molitor larvae which significantly (P < 0.05) decreased the mortality in control larvae but no significant change in SPE silenced larvae (Fig 6B).This suggested that DSP1 is dependent on SPE for immune mediation.This finding concludes that DSP1 depend on SPE to follow the Toll-spa ¨tzle pathway to induce the immune activation.

Discussion
Dorsal switch protein 1 (DSP1), an insect homolog of vertebrate HMGB1 protein was characterized as a co-repressor of Dorsal protein in Drosophila [7].Dorsal is one of the x 10 4 cells/larva) were injected to L6 larvae of T. molitor already (24 h before) injected with dsSPE and dsCON (1 μg/ larva).dsCON was made from a viral gene CpBV302.For immune mediation test of rDSP1, at 24 h post injection of transcriptional activators from the Rel/NF-kB family [7].In Drosophila, DSP1 also play role in embryo development, differentiation, and segmentation [6,30,31].In mosquito, Aedes aegypti, another HMGB1 having high homologies with mammalian HMGB1 and Drosophila DSP1 [32] has been identified.This mosquito HMGB1 facilitate the binding of transcriptional factor 'Rel' to the nuclear factor kappa B (NF-kB) promoter for antiviral gene expression against a dengue viral infection [33].A lepidopteran insect (Plodia interpunctella), apart from dipterans, also possesses HMGB1-like proteins [34].Recent study revealed the DAMP role of DSP1 in lepidopteran S. exigua [11], coleopteran T. molitor [12] and dipteran Aedes albopictus [35] that mediate different immune responses like PLA 2 activity, nodulation formation, AMP synthesis, PO activity etc. [11][12][13].Subsequent study in S. exigua reports that DSP1 uses Tollspa ¨tzle pathway for immune mediation [14].But we do not know the role of SPE in this pathway.To address this, we coined this study in T. molitor to know the involvement of SPE in DSP1 induced immune mediation through Toll-spatzle pathway.For this, recombinant DSP1 was injected into the larvae to observe gain of function or additive immune responses and lose of function after RNAi of SPE.

Fig 3 .
Fig 3. Role of SPE for the induction of immune responses by E. mundtii and rDSP1.(A) RNAi efficiencies of Tm-SPE gene by injecting gene-specific dsRNA ('dsSPE', 1 μg/larva) to L6 larvae.A viral gene, CpBV302, was used as a control dsRNA ('dsCON').(B) E. mundtii and rDSP1 induced mediation of hemocyte nodulation, (C) PO activity and (D) Pictorial representation of change in plasma due of PO activity.L6 larvae of T. molitor were injected with E. mundtii (2.2 x 10 5 cells/larvae) dissolved in PBS or E. mundtii (2.2 x 10 5 cells/larvae) along with recombinant DSP1 ('rDSP1, 0.6 μg/larva).At 8 h of E. mundtii or rDSP1 injection, nodules were counted in each larva by dissecting and plasma was collected for PO activity analysis.Naïve was injected with PBS.Knock-down of Tm-SPE expression was done by injecting dsRNA specific to Tm-SPE (1 μg/larva) into L6 larvae before 24 h of E. mundtii or rDSP1 injection.A viral gene, CpBV302, was used as a control dsRNA ('dsCON').Each treatment was replicated three times.Asterisks indicate significant differences between dsSPE and dsCON treated group at Type I error = 0.05 (LSD test).Different letters above standard deviation bars denotes significant difference among means at Type I error = 0.05 (LSD test).